PROTON NMR INVESTIGATION OF THE [4FE-4S](1+) CLUSTER ENVIRONMENT OF NITROGENASE IRON PROTEIN FROM AZOTOBACTER-VINELANDII - DEFINING NUCLEOTIDE-INDUCED CONFORMATIONAL-CHANGES

被引:31
作者
LANZILOTTA, WN [1 ]
HOLZ, RC [1 ]
SEEFELDT, LC [1 ]
机构
[1] UTAH STATE UNIV,DEPT CHEM & BIOCHEM,LOGAN,UT 84322
关键词
D O I
10.1021/bi00048a007
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
This work presents the complete assignment of the isotropically shifted H-1 NMR resonances of Azotobacter vinelandii nitrogenase iron protein (Fe protein) to beta-CH2 and alpha-CH protons of the [4Fe-4S](1+) cluster cysteinyl ligands. Four resonances were observed for the reduced Fe protein with chemical shifts of 49, 23, 17, and 13 ppm. T-l measurements and analysis of relative peak areas coupled with one-dimensional nuclear Overhauser effect (NOE) difference spectra were used to assign the two most downfield-shifted resonances (49 and 23 ppm) to cysteinyl ligand beta-CH2 protons and the 17 and 14 ppm resonances to cysteinyl ligand alpha-CH protons. Temperature dependence studies of the isotropically shifted protons revealed both Curie and anti-Curie behavior. These results, along with previous Mossbauer studies of the Fe protein, allowed the assignment of signal A (49 ppm) to four beta-CH2 protons and signal C (17 ppm) to 2 alpha-CH protons of two cysteinyl ligands bound to a mixed-valence iron pair (Fe3+-Fe2+) of the [4Fe-4S](1+) cluster. Signal B (23 ppm) was assigned to four beta-CH2 protons, and signal C (17 ppm) and D (13 ppm) were assigned to two alpha-CH protons of two cysteinyl ligands bound to a ferrous pair of irons (2Fe(2+)). The effects of MgATP, MgADP, and Mg-adenosine-beta, gamma-methylene-5'-triphosphate binding to the Fe protein on the assigned resonances were established and are discussed in the context of nucleotide-induced changes in the protein environment of the [4Fe-4S] cluster. In addition, conditions are described that prevent the long-standing problem of A, vinelandii Fe protein self-oxidation.
引用
收藏
页码:15646 / 15653
页数:8
相关论文
共 46 条
  • [1] PROPERTIES OF CUPRIC IONS IN BENZYLAMINE OXIDASE FROM PIG PLASMA AS STUDIED BY MAGNETIC-RESONANCE AND KINETIC METHODS
    BARKER, R
    BODEN, N
    CAYLEY, G
    CHARLTON, SC
    HENSON, R
    HOLMES, MC
    KELLY, ID
    KNOWLES, PF
    [J]. BIOCHEMICAL JOURNAL, 1979, 177 (01) : 289 - 302
  • [2] H-1-NMR STUDIES ON PARTIALLY AND FULLY REDUCED 2(4FE-4S) FERREDOXIN FROM CLOSTRIDIUM-PASTEURIANUM
    BERTINI, I
    BRIGANTI, F
    LUCHINAT, C
    MESSORI, L
    MONNANNI, R
    SCOZZAFAVA, A
    VALLINI, G
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1992, 204 (02): : 831 - 839
  • [3] NUCLEAR-MAGNETIC-RESONANCE OF PARAMAGNETIC METALLOPROTEINS
    BERTINI, I
    TURANO, P
    VILA, AJ
    [J]. CHEMICAL REVIEWS, 1993, 93 (08) : 2833 - 2932
  • [4] THE EFFECT OF THE REDOX POTENTIAL ON THE ACTIVITY OF THE NITROGENASE AND ON THE FE-PROTEIN OF AZOTOBACTER-VINELANDII
    BRAAKSMA, A
    HAAKER, H
    GRANDE, HJ
    VEEGER, C
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1982, 121 (03): : 483 - 491
  • [5] BURRIS RH, 1991, J BIOL CHEM, V266, P9339
  • [6] NMR SPECTROSCOPIC STUDIES OF PARAMAGNETIC PROTEINS - IRON-SULFUR PROTEINS
    CHENG, H
    MARKLEY, JL
    [J]. ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE, 1995, 24 : 209 - 237
  • [7] CHROMY V, 1974, CLIN CHEM, V20, P1362
  • [8] NITROGENASE METALLOCLUSTERS - STRUCTURES, ORGANIZATION, AND SYNTHESIS
    DEAN, DR
    BOLIN, JT
    ZHENG, LM
    [J]. JOURNAL OF BACTERIOLOGY, 1993, 175 (21) : 6737 - 6744
  • [9] H-1-NMR INVESTIGATION OF THE ELECTRONIC-STRUCTURE OF THE 4-IRON FERREDOXIN FROM THE HYPERTHERMOPHILIC ARCHAEON THERMOCOCCUS-LITORALIS
    DONAIRE, A
    GORST, CM
    ZHOU, ZH
    ADAMS, MWW
    LAMAR, GN
    [J]. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1994, 116 (15) : 6841 - 6849
  • [10] COMPLEMENTARY FUNCTIONING OF COMPONENT PROTEINS OF NITROGENASE FROM SEVERAL BACTERIA
    EMERICH, DW
    BURRIS, RH
    [J]. JOURNAL OF BACTERIOLOGY, 1978, 134 (03) : 936 - 943